Journal of Dairy Research (2014) 81 82–90. doi:10.1017/S0022029913000691
© Proprietors of Journal of Dairy Research 2014
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Effect of feeding linseed oil in diets differing in forage to concentrate ratio: 1. Production performance and milk fat content of biohydrogenation intermediates of α-linolenic acid Leacady Saliba, Rachel Gervais, Yolaine Lebeuf and P. Yvan Chouinard* Département des sciences animales, Université Laval, Québec, Québec G1V 0A6, Canada Received 25 February 2013; accepted for publication 31 October 2013
To evaluate the interaction between the levels of dietary concentrate and linseed oil (LO) on milk fatty acid (FA) profile, 24 Holstein cows were used in a randomised complete block design based on days in milk, with a 2 × 2 factorial arrangement of treatments. Within each block, cows were fed one of four experimental diets containing 30% concentrate (LC) or 70% concentrate (HC), without LO (NLO) or with LO supplemented at 3% of dietary dry matter. Milk FA profiles were analysed with a special emphasis on the intermediates of the predominant trans-11, and a putative trans-13 pathways of ruminal biohydrogenation of cis-9, cis-12, cis-15 18:3. Feeding LO increased the concentrations of cis-9, cis-12, cis-15 18:3 and trans-11, cis-15 18:2 in milk fat, and these increases were of a higher magnitude when LO was added in HC as compared with LC diet (interaction of LO by concentrate). A treatment interaction was also observed for the level of trans-11 18:1 which was higher when feeding LO, but for which the increase was more pronounced with the LC as compared with HC diet. The concentrations of cis-15 18:1 and cis-9, trans-11, cis-15 18:3 were higher in cows fed LO, but feeding HC diets decreased milk fat content of cis-15 18:1 and a tendency for a decrease in cis-9, trans-11, cis15 18:3 was apparent. Feeding LO increased milk fat content of trans-13 18:1 and cis-9, trans-13 18:2, while the concentrations of these two isomers were not affected by the level of dietary concentrates. The isomer cis-9, trans-13, cis-15 18:3 has not been detected in any of the milk samples. In conclusion, interactions were observed between LO and dietary concentrates on the proportions of some intermediates of the trans-11 biohydrogenation pathway. The presence of trans-13 18:1 and cis-9, trans-13 18:2 supports the existence of a trans-13 pathway, but an 18:3 intermediate with a trans-13 double bond has not been identified. Keywords: Dairy cow, flaxseed, biohydrogenation, forage to concentrate ratio.
Full-fat linseed and linseed oil, which are sources of α-linolenic acid (cis-9, cis-12, cis-15 18:3), have been investigated for their effects on lactation (Petit, 2010) and reproductive (Petit et al. 2002) performance, FA profile and physical properties of milk fat (Hurtaud et al. 2010), and enteric methane emission (Martin et al. 2008) in dairy cows. When cows receive full-fat linseed or linseed oil, the ruminal biohydrogenation of cis-9, cis-12, cis-15 18:3 produces a large number of FA isomers (Loor et al. 2004; Flachowsky et al. 2006). These biohydrogenation intermediates can be absorbed and incorporated in milk fat of lactating animals, either directly, or following their metabolic transformation in body tissues (e.g. desaturation and elongation).
*For correspondence; e-mail: [email protected]
Dietary factors affecting ruminal biohydrogenation of linoleic acid (cis-9, cis-12 18:2) have been extensively evaluated (Bauman & Griinari, 2001). More specifically, increased production of trans-10 18:1 is observed when feeding high doses of unsaturated FA with low-fibre diets. However, less is known about the effects of diet on different pathways of ruminal biohydrogenation of cis-9, cis-12, cis15 18:3. The predominant pathway in the biohydrogenation of cis-9, cis-12, cis-15 18:3 involves an initial isomerisation where the cis-12 bond is converted to a trans-11 bond to produce cis-9, trans-11, cis-15 18:3 (Harfoot, 1981). The second and third steps consist of the hydrogenation of cis-9 and cis-15 bonds to produce trans-11, cis-15 18:2 and trans11 18:1, respectively. After absorption, trans-11 18:1 can be desaturated to cis-9, trans-11 18:2 by the action of the Δ9 desaturase (Griinari et al. 2000). Destaillats et al. (2005)
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Table 1. Ingredients and chemical composition of experimental diets (g 100/g dry matter) Treatment† NLO Pretreatment
LC
LO HC
LC
HC
Ingredient, g/100 g of dry matter Corn silage Grass/Legume silage‡ Grass hay Rolled barley Cracked corn Soybean meal Corn gluten meal Linseed oil Vitamins and minerals
28·6 23·4 6·9 11·9 11·5 11·0 4·5 — 2·2
Dry matter, g/100 g Crude protein Starch ADF NDF NEL, Mcal/kg dry matter
47·8 15·0 30·1 20·1 33·1 1·61
14:0 16:0 cis-9 16:1 18:0 cis-9 18:1 cis-11 18:1 cis-9, cis-12 18:2 cis-9, cis-12, cis-15 18:3 20:0 Total fatty acids
60·0 25·0 60·0 10·0 5·0 10·0 9·0 28·6 9·0 9·0 28·6 5·4 6·7 7·5 7·3 3·3 3·3 3·3 — — 3·0 2·0 2·0 2·0 Chemical composition, g/100 g of dry matter 45·8 62·9 45·9 15·4 15·3 14·9 13·2 37·5 10·5 29·0 16·3 29·1 45·3 27·3 45·9 1·43 1·56 1·54 Fatty acid profile, g/100 g of dry matter 0·12 0·11 0·11 4·18 4·48 5·00 0·07 0·07 0·08 0·50 0·50 1·52 2·34 4·25 7·72 0·20 0·20 0·42 8·29 12·26 11·84 7·31 4·89 19·56 0·15 0·14 0·16 23·16 26·89 46·43
25·0 5·0 28·6 25·0 8·1 3·3 3·0 2·0 64·5 15·6 35·6 18·4 25·8 1·65 0·09 5·46 0·07 1·55 9·86 0·46 16·55 16·98 0·15 51·15
† NLO = no linseed oil; LO = with linseed oil, LC = low concentrate; HC = high concentrate ‡ Grass/legume silage was constituted of 11% ladino clover, 34% timothy, and 55% bromegrass
proposed an alternative pathway in the biohydrogenation of cis-9, cis-12, cis-15 18:3 involving an initial isomerisation where the cis-12 bond is converted to a trans-13 bond, leading to the sequential production of cis-9, trans-13, cis-15 18:3, cis-9, trans-13 18:2 or trans-13, cis-15 18:2, and trans-13 18:1. As for several octadecenoic acids, trans-13 18:1 could be desaturated after absorption to produce cis-9, trans-13 18:2 (Destaillats et al. 2005). In support of the pathway proposed by Destaillats et al. (2005), few experiments have shown the presence of trace amounts of cis-9, trans-13, cis-15 18:3 in meat and milk fat from ruminants (Plourde et al. 2007; Rego et al. 2009). As opposed to cis-9, cis-12 18:2, the effects of diet on the different biohydrogenation pathways of cis-9, cis-12, cis-15 18:3 are not well documented. The objective of this experiment was therefore to evaluate the effect of feeding linseed oil in diets differing in forage to concentrate ratio (F:C) on milk yield and composition, and on milk FA profile. It was hypothesised that supplying linseed oil in different basal diets affects the relative importance of different pathways involved in ruminal biohydrogenation of
cis-9, cis-12, cis-15 18:3, resulting in varying concentrations of specific FA intermediates in milk fat.
Materials and methods Animals and diets The experiment was conducted at the Centre de Recherche en Sciences Animales de Deschambault (Deschambault, QC, Canada). All procedures involving dairy cows followed the regulations of the Canadian Council on Animal Care (1993), and were approved by the Université Laval animal care committee. Twenty-four lactating Holstein cows (637 ± 68 kg body weight; 168 ± 53 d in milk; 9 primiparous and 15 multiparous) housed in a tie-stall barn were fed a pretreatment total mixed ration based on grass/legume silage and corn silage (Table 1). Cows were then assigned to a randomised complete block design based on days in milk with a 2 × 2 factorial arrangement of treatments. Within each block, cows were fed one of four experimental diets
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containing 30% concentrate (LC) or 70% concentrate (HC), without linseed oil (NLO) or with linseed oil (LO) supplemented at 3% of dietary dry matter (Table 1). The treatment period lasted 4 weeks. Throughout the experiment, diets were offered to the cows once daily as a total mixed ration, and the amount of feed offered was adjusted to ensure 10% refusals. Free access to water was provided at all time. Sampling, measurements and analyses A first sampling and measurement phase was conducted during the last week of the pretreatment period (week 0), and a second during the last week of the treatment period (week 4). At each of those phases, cows were weighed for 3 consecutive days following the a.m. milking. Dry matter intake was recorded and feed samples were collected for 3 consecutive days and pooled by treatment. A first subsample was dried at 55 °C for 3 days to determine dry matter intake. All rations were analysed by wet chemistry (Dairy One Laboratory, Ithaca, NY, USA) according to the following methods: dry matter (method 2.2.1.1; National Forage Testing Association; Undersander et al. 1993), residual moisture (method 991.01; AOAC International, 2012), ADF (ANKOM Technology method 5: ADF in feeds – filter bag technique for A200; solutions as in method 973.18; AOAC International, 2012), NDF (ANKOM Technology method 6: NDF in feeds – filter bag technique for A200; solutions as in Van Soest et al. 1991), and starch (application note number 319; YSI Inc. Life Sciences, Yellow Springs, Ohio). After freeze drying and grinding through a 1-mm screen, FA profile of total mixed rations was determined. Fatty acids were directly transesterified, and FA methyl esters were extracted following the method described by Sukhija & Palmquist (1988) with modifications. In the modified procedure, benzene was replaced by toluene and incubation in 3 ml HCl 5% (v/v) was performed at 50 °C for 30 min and followed a prior incubation in 3 ml sodium methoxide (0·5 M in methanol) at 70 °C for 60 min. Cows were milked at 0700 and 1700 h. Milk was sampled, and milk yield was recorded for 3 consecutive days. Daily milk composites were made from p.m. and a.m. milk samples proportionally to milk yield. A first subsample was preserved with bronopol and kept at 4 °C before analyses of fat, protein, and lactose using a Foss Milkoscan 4000 (Foss Electric, Hillerød, Denmark) combined with a Bentley 2000 (Bentley Instruments, Chaska, MN) at Valacta (Dairy Production Centre of Expertise, Ste-Anne-deBellevue, Québec, Canada). A second subsample was stored at 20 °C without preservative for FA analysis. Before lipid extraction for FA analysis, milk samples were thawed in water at 36 °C for 30 min. Milk fat was then extracted and FA were methylated according to Chouinard et al. (1997). Milk FA profile was determined using a gas chromatograph (Agilent 7890A; Agilent Technologies, Santa Clara, CA, USA) equipped with a 100-m CP-Sil-88 capillary column (0·25 μm i.d., 0·20 μm film thickness; Agilent
Technologies Canada Inc., Mississauga, Canada), and a flame ionisation detector. In order to screen milk FA composition, a first temperature programme was used where, at the time of injection, the column temperature was 80 °C for 1 min., then ramped at 2 °C/min. to 215 °C and maintained for 21·5 min. Two additional isothermal temperature programmes (150 and 175 °C; Kramer et al. 2008) were used to separate isomers that were co-eluting in the first programme. For the separation and quantification of cis-9, trans-11, cis-15 18:3 and cis-9, trans-13, cis-15 18:3, a fourth temperature programme was used where at the time of sample injection, the column temperature was 120 °C for 180 min., then increased at 10 °C/min. to 220 °C and maintained isothermal for 20 min. (Plourde et al. 2007). For the separation and quantification of trans-13 18:1 and trans14 18:1, trans FA methyl esters were first isolated using silver ion-thin layer chromatography followed by a GC analysis of this fraction with a fifth programme at a low isothermal temperature of 120 °C (Kramer et al. 2008). Most FA peaks were identified and quantified using either a quantitative mixture or pure methyl ester standards (Larodan Fine Chemicals, Malmö, Sweden; Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada; Matreya LLC, Pleasant Gap, PA, USA; Nu Chek Prep, Elysian, MN, USA; Naturia, Sherbrooke, QC, Canada). Standards for isomers that were not available commercially were identified by order of elution according to Precht et al. (2001) and Kramer et al. (2008). Statistical analysis Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) with two models depending on type of data collected. The first model was used for body weight, dry matter intake, milk yield, and milk composition. This model included data collected during the last week of the pre-trial period as covariates, and also level of concentrate, oil supplementation, and their interaction as fixed effects. Block was included as a random effect. Because data on milk FA profile from the pre-trial period were not available, a second model was used to determine treatment effects on milk FA composition which included the fixed effects of level of concentrate, oil supplementation, and their interaction, and block as a random effect. Results are reported as least square means and standard errors of the means. Differences between treatments were declared at P 4 0·05, and tendencies from P > 0·05 to P < 0·10.
Results Dietary concentrations of crude protein were similar between treatments (Table 1). Both HC diets (NLO and LO) contained about 40% more dry matter, three times more starch, and 40% less ADF and NDF than both LC diets (NLO and LO). The HC diets which contained higher concentration of cracked corn and rolled barley had 40% more cis-9
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Table 2. Body weight, dry matter intake (DMI), milk yield, and milk composition in cows fed high (HC) or low (LC) concentrate diets without supplemental oil (NLO), or supplemented at 3% of dry matter with linseed oil (LO) Treatment NLO
LO
Item
LC
HC
Body weight, kg DMI, kg/d Milk yield, kg/d
648 21·5 25·4
663 25·6 32·2
Fat Protein Lactose
4·22 3·33 4·60
Fat Protein Lactose
1,046 836 1,174
P-value†
LC
654 19·6 26·9 Composition, g/100 g 3·76 4·07 3·59 3·12 4·72 4·58 Yield, kg/d 1,165 1,106 1,136 829 1,525 1,236
HC
SEM
LO
C
LO × C
653 23·9 34·1
6 0·8 1·2
0·75 0·03 0·12
0·22 < 0·01 < 0·01
0·21 0·90 0·87
3·37 3·42 4·80
0·09 0·03 0·04
< 0·01 < 0·01 0·40
< 0·01 < 0·01 < 0·01
0·18 0·50 0·18
1,160 1,160 1,640
48 40 58
0·54 0·82 0·11
0·07 < 0·01 < 0·01
0·46 0·68 0·64
† P-value for the effects of linseed oil (LO), concentrate (C), and the interaction of linseed oil by concentrate (LO × C)
Table 3. Fatty acid intake in cows fed high (HC) or low (LC) concentrate diets without supplemental oil (NLO), or supplemented at 3% of dry matter with linseed oil (LO) Treatment NLO
LO
P-value†
Fatty acid, g/day
LC
HC
LC
HC
SEM
LO
C
14:0 16:0 cis-9 16:1 18:0 cis-9 18:1 cis-11 18:1 cis-9, cis-12 18:2 cis-9, cis-12, cis-15 18:3 20:0 Total fatty acids
2·6 90·0 1·5 10·9 51·0 4·3 179·0 158·1 3·2 500·5
2·8 114·3 1·8 12·4 106·7 5·0 312·4 120·0 3·6 678·6
2·2 97·6 1·6 29·8 150·0 8·2 228·6 385·8 3·1 907·2
2·2 130·0 1·7 36·8 233·8 10·9 393·1 403·7 3·6 1215·3
0·1 4·6 0·1 1·4 8·4 0·4 13·8 15·1 0·1 43·4
< 0·01 0·01 0·65 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·73 < 0·01
0·13 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·46 < 0·01 < 0·01
LO × C 0·11 0·35 0·12 0·04 0·09 0·01 0·24 0·06 0·71 0·12
† P-value for the effects of linseed oil (LO), concentrate (C), and the interaction of linseed oil by concentrate (LO × C)
18:1 and cis-9, cis-12 18:2, and 19% less cis-9, cis-12, cis-15 18:3 compared with LC diets. The addition of LO doubled the concentration of total FA in the diet compared with NLO treatments. Moreover, adding LO to diets increased the proportions of 18:0, cis-9 18:1, and cis-9, cis-12, cis-15 18:3 by about 3-fold as compared with NLO. No interaction of oil by concentrate has been observed for animal performance and concentration of major milk constituents (Table 2). Final body weight after the 4-wk experimental period was similar among treatments. Feeding HC diets increased dry matter intake, milk yield, as well as protein and lactose content and yield (P < 0·01). Adding LO decreased dry matter intake and milk protein content, but increased intake of cis-9, cis-12 18:2 and cis-9, cis-12, cis-15 18:3 (Table 3). Fat content decreased with LO
supplementation, as well as with high concentrate diets (P < 0·01), while fat yield tended to be higher with HC compared with LC (P = 0·07). Feeding LO increased the concentration of cis-9, cis-12, cis-15 18:3 in milk fat (Table 4), and this increase was of a higher magnitude when LO was added in HC as compared with LC diet (interaction of LO by concentrate P < 0·01). The concentration of cis-9, cis-12 18:2 was not affected by LO, but was increased when feeding HC as compared with LC diets. A treatment interaction was observed for the level of trans11 18:1 which was increased by feeding LO, but for which the increase was more pronounced with the LC as compared with HC diets. Milk fat concentrations of trans-15 18:1 and trans-11, cis-15 18:2 were also higher in milk of cows fed LO,
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Table 4. Milk fat composition in cows fed high (HC) or low (LC) concentrate diets without supplemental oil (NLO), or supplemented at 3% of dry matter with linseed oil (LO) Treatment NLO Item, g/100 g Saturated 4:0 5:0 6:0 7:0 8:0 9:0 10:0 11:0 12:0 iso 13:0 anteiso 13:0 13:0 iso 14:0 14:0 iso 15:0 anteiso 15:0 15:0 iso 16:0 16:0 iso 17:0‡ anteiso 17:0§ 17:0 iso 18:0 18:0 19:0 20:0 22:0 24:0 Monounsaturated cis-9 10:1 trans 12:1¶ cis-9 12:1 cis-9 14:1 cis-11 14:1 trans 15:1¶ cis-9 15:1 trans-4 16:1 trans-5 16:1 trans-6-7 16:1 trans-8 16:1 trans-9 16:1 cis-7 16:1 + trans-11-12 16:1 cis-9 16:1 + trans-13 16:1 cis-11 16:1 cis-12 16:1 + trans-14 16:1 cis-13 16:1 cis-14 16:1 cis-7 17:1 cis-8 17:1 cis-9 17:1 trans-4 18:1 trans-5 18:1 trans-6-8 18:1
LO
P-value†
LC
HC
LC
HC
SEM
LO
C
LO × C
2·935 0·024 1·818 0·028 1·074 0·030 2·627 0·049 3·132 0·024 0·007 0·082 0·090 10·662 0·219 0·401 1·129 0·197 30·540 0·258 0·354 0·484 0·046 7·398 0·038 0·147 0·060 0·039
2·355 0·033 1·540 0·042 1·028 0·057 3·048 0·130 3·967 0·024 0·005 0·177 0·054 11·615 0·173 0·379 1·418 0·162 27·473 0·286 0·362 0·491 0·032 7·366 0·052 0·121 0·037 0·025
2·908 0·016 1·502 0·015 0·756 0·014 1·572 0·015 1·721 0·023 0·007 0·043 0·074 7·715 0·172 0·328 0·785 0·143 19·555 0·258 0·303 0·400 0·046 12·380 0·010 0·197 0·055 0·032
2·352 0·021 1·338 0·021 0·827 0·021 2·074 0·044 2·496 0·027 0·006 0·081 0·045 9·586 0·150 0·339 0·846 0·210 19·951 0·301 0·342 0·364 0·029 11·625 0·013 0·170 0·039 0·021
0·157 0·002 0·090 0·004 0·061 0·006 0·150 0·013 0·150 0·003 0·001 0·015 0·006 0·293 0·012 0·021 0·082 0·023 1·019 0·012 0·018 0·017 0·003 0·607 0·004 0·008 0·004 0·003
0·93 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·71 0·63 < 0·01 0·05 < 0·01 < 0·01 0·02 < 0·01 0·86 < 0·01 0·50 0·04 < 0·01 0·61 < 0·01 < 0·01 < 0·01 0·58 0·02
< 0·01 < 0·01 0·02 0·02 0·84 0·01 < 0·01 < 0·01 < 0·01 0·50 0·03 < 0·01 < 0·01 < 0·01 0·01 0·78 < 0·05 0·37 0·12 < 0·01 0·15 0·38 < 0·01 0·52 0·08 < 0·01 < 0·01 < 0·01
0·94 0·39 0·53 0·30 0·35 0·12 0·79 < 0·05 0·84 0·53 0·81 0·06 0·57 0·12 0·32 0·45 0·18 0·01 < 0·05 0·51 0·33 0·21 0·58 0·56 0·21 0·90 0·38 0·41
0·261 0·068 0·078 1·025 0·044 0·024 0·013 0·005 0·006 0·012 0·023 0·055 0·133 1·229 0·027 0·073 0·205 0·016 0·030 0·043 0·161 0·006 0·007 0·140
0·230 0·077 0·103 1·008 0·070 0·016 0·011 0·005 0·008 0·010 0·021 0·030 0·160 1·202 0·029 0·040 0·228 0·014 0·028 0·019 0·161 0·007 0·009 0·161
0·146 0·029 0·035 0·628 0·019 0·020 0·012 0·006 0·007 0·006 0·025 0·098 0·189 0·863 0·018 0·110 0·093 0·008 0·023 0·035 0·144 0·025 0·019 0·336
0·171 0·042 0·053 0·782 0·035 0·013 0·009 0·004 0·006 0·007 0·021 0·044 0·182 0·802 0·020 0·064 0·114 0·008 0·020 0·016 0·118 0·012 0·014 0·307
0·012 0·004 0·004 0·053 0·003 0·002 0·001 0·001 0·001 0·001 0·002 0·006 0·008 0·094 0·002 0·007 0·009 0·001 0·001 0·001 0·013 0·001 0·001 0·019
< 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·03 0·11 0·48 0·88 < 0·01 0·35 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·03 < 0·01 < 0·01 < 0·01
0·80 < 0·01 < 0·01 0·17 < 0·01 < 0·01 0·03 0·11 0·77 0·74 0·04 < 0·01 0·18 0·64 0·36 < 0·01 0·02 0·44 < 0·01 < 0·01 0·31 < 0·01 0·07 0·82
0·02 0·55 0·44 0·09 0·09 0·91 0·37 0·16 0·08 0·10 0·38 0·02 0·04 0·85 0·74 0·39 0·92 0·53 0·48 < 0·01 0·32 < 0·01 < 0·01 0·20
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Table 4. (Cont.) Treatment NLO
LO
P-value†
Item, g/100 g
LC
HC
LC
HC
SEM
LO
trans-9 18:1 trans-10 18:1 trans-11 18:1 trans-12 18:1 trans-13 18:1 trans-14 18:1 trans-15 18:1 trans-16 18:1 cis-6-8 18:1 cis-9 18:1†† cis-11 18:1 cis-12 18:1 cis-13 18:1 cis-14 18:1 cis-15 18:1 cis-9 20:1 cis-11 20:1 Nonconjugated 18:2 trans-9, trans-12 18:2 trans-8, cis-12 18:2 trans-8, cis-13 18:2 trans-9, cis-12 18:2 trans-11, cis-15 18:2 cis-9, trans-12 18:2 cis-9, trans-13 18:2 cis-9, cis-12 18:2 Conjugated 18:2 cis-9, trans-11 18:2‡‡ trans-10, cis-12 18:2 Other unsaturated cis-6, cis-9, cis-12 18:3 cis-9, cis-12, cis-15 18:3 cis-9, trans-13, cis-15 18:3 cis-9, trans-11, cis-15 18:3 cis-11, cis-14 20:2 cis-8, cis-11, cis-14 20:3 cis-11, cis-14, cis-17 20:3 cis-5, cis-8, cis-11, cis-14 20:4 cis-8, cis-11, cis-14, cis-17 20:4 cis-5, cis-8, cis-11, cis-14, cis-17 20:5 cis-13, cis-16 22:2 cis-7, cis-10, cis-13, cis-16 22:4 cis-4, cis-7, cis-10, cis-13, cis-16 22:5 cis-7, cis-10, cis-13, cis-16, cis-19 22:5 cis-4, cis-7, cis-10, cis-13, cis-16, cis-19 22:6 Other fatty acids Glycerol
0·117 0·123 1·130 0·122 0·074 0·116 0·212 0·192 0·151 13·183 0·352 0·169 0·079 0·036 0·036 0·096 0·018
0·127 0·182 0·637 0·177 0·110 0·170 0·166 0·216 0·116 14·271 0·741 0·230 0·086 0·033 0·023 0·094 0·021
0·288 0·306 2·176 0·563 0·526 0·879 0·456 0·719 0·164 19·283 0·849 0·634 0·129 0·100 0·085 0·131 0·027
0·236 0·562 1·002 0·498 0·464 0·741 0·556 0·634 0·082 19·711 0·804 0·410 0·116 0·066 0·064 0·141 0·027
0·016 0·066 0·106 0·032 0·035 0·058 0·036 0·034 0·012 0·755 0·048 0·025 0·012 0·007 0·007 0·007 0·003
< 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·34 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01
0·20 0·03 < 0·01 0·89 0·71 0·48 0·39 0·38 < 0·01 0·28 < 0·01 < 0·01 0·77 0·01 < 0·01 0·56 0·62
0·07 0·15 < 0·01 0·07 0·18 0·11 0·03 0·13 0·06 0·63 < 0·01 < 0·01 0·33 0·03 0·42 0·42 0·55
0·047 0·105 0·052 0·019 0·164 0·017 0·094 1·181
0·025 0·162 0·063 0·019 0·049 0·023 0·122 1·921
0·157 0·299 0·212 0·069 0·880 0·060 0·261 1·240
0·063 0·300 0·233 0·035 0·359 0·056 0·235 1·670
0·011 0·023 0·017 0·006 0·048 0·006 0·019 0·097
< 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·25
< 0·01 0·22 0·37 0·01 < 0·01 0·94 0·93 < 0·01
< 0·01 0·25 0·76 0·01 < 0·01 0·43 0·16 0·07
0·515 0·002
0·359 0·003
0·879 0·004
0·510 0·004
0·063 0·0005
< 0·01 0·04
< 0·01 0·23
0·11 0·17
0·026 0·377 nd 0·025 0·028 0·080 0·008 0·108 0·006 0·045 0·041 0·020 0·015 0·051 0·007 0·825 12·360
0·031 0·298 nd 0·021 0·027 0·109 0·007 0·146 0·007 0·034 0·021 0·029 0·016 0·055 0·009 0·718 12·224
0·019 0·463 nd 0·050 0·022 0·058 0·013 0·084 0·007 0·036 0·033 0·019 0·011 0·056 0·009 1·913 11·891
0·017 0·550 nd 0·037 0·019 0·063 0·010 0·084 0·008 0·037 0·020 0·019 0·017 0·060 0·008 1·419 11·851
< 0·01 < 0·01 — < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·08 0·08 < 0·01 0·02 0·43 0·18 0·75 < 0·01 < 0·01
0·35 0·91 — 0·06 0·25 < 0·01 0·20 0·01 0·03 0·02 < 0·01 0·03 0·12 0·32 0·77 0·02 0·24
0·03 0·01 — 0·31 0·38 0·02 0·35 0·02 0·45 < 0·01 0·06 0·04 0·21 0·97 0·41 0·10 0·52
0·001 0·030 — 0·004 0·002 0·005 0·001 0·008 0·001 0·002 0·002 0·003 0·002 0·004 0·002 0·113 0·073
† P- value for the effects of linseed oil (LO), concentrate (C), and the interaction of linseed oil by concentrate (LO × C) ‡ Coelution with minor concentration of trans-10 16:1 § Coelution with minor concentration of cis-10 16:1 ¶ Position of the double bound was not confirmed †† Coelution with minor concentration of cis-10 18:1 ‡‡ Coelution with trans-7, cis-9 18:2
C
LO × C
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but the increase was of a lower magnitude when LO was added in LC compared with HC diets. Feeding LO increased milk fat content of trans-13 18:1 and cis-9, trans-13 18:2, while their concentrations were not affected by dietary concentrates (Table 4). The isomer cis-9, trans-13, cis-15 18:3 has not been detected in any of the milk samples. The concentrations of cis-15 18:1, cis-9, trans-11 18:2 and cis-9, trans-11, cis-15 18:3 were higher in milk of cows fed LO, but feeding HC diets decreased milk fat content of these three FA without interaction between dietary treatments. Dietary LO increased the concentrations of trans-10 18:1 and trans-10, cis-12 18:2 in milk fat; and the proportion of trans-10 18:1 was further increased by feeding HC as compared with LC. Finally, the level of milk 18:0 was increased by feeding LO, but was not affected by dietary concentrates.
Discussion Feeding LO, and supplying more concentrate, increased the dietary proportions of FA and starch, and therefore improved the energy density of the diets (Table 1). As observed in the current trial, Sterk et al. (2011) also reported that shifting from a high forage (65:35 F:C) to a high concentrate (35:65 F:C) diet increased dry matter intake and milk yield in dairy cows. The higher dry matter intake with HC diets is in agreement with a lower dietary NDF content as suggested by Allen (2000). The observed increase in milk yield with HC could then be explained by a greater intake of dry matter, with a higher nutrient density. Supplementing dairy diets with LO, at similar or lower concentrations than the 3% level used in the current trial, has been shown to have no effect on dry matter intake (Loor et al. 2005; Flachowsky et al. 2006; Benchaar et al. 2012). However, Chilliard et al. (2009) reported a lower dry matter intake with higher levels of LO supplementation (5·7% of dry matter) using corn silage based diets. Feeding HC diets, with higher starch content, increased milk protein concentration and yield. Sterk et al. (2011) observed similar results when supplying a greater amount of starch, a glucogenic precursor which has been shown by these authors to be correlated with milk protein synthesis. Also, in a review by Emery (1978), a positive correlation was established between milk protein concentration and energy intake when concentrate was substituted for forage. The effects observed in the current trial on milk composition are consistent with previously published results showing lower milk fat content when feeding high concentrate diets (Griinari et al. 1998; Sterk et al. 2011). However, the decrease in milk fat content was more than compensated by an increase in milk production, so that a tendency for a higher fat yield was observed with HC diets in the present experiment. Dietary addition of LO increased the intake of cis-9, cis-12, cis-15 18:3, and this increase tended to be of a higher
magnitude with HC as compared with LC (P = 0·06). These variations were reflected in milk fat, where concentrations of cis-9, cis-12, cis-15 18:3 were increased when cows received LO diets with the highest concentration being observed with HCLO. Similarly, feeding HC diets increased the intake of cis-9, cis-12 18:2 which explained the higher concentration of this essential FA in milk fat as compared with LC diets. Among the intermediates of ruminal biohydrogenation, the treatment interaction observed on the level of trans-11, cis-15 18:2, that was increased by feeding LO, but for which the increase was more pronounced with the HC as compared with LC, is consistent with a similar interaction observed in the supply of its precursor e.g., dietary cis-9, cis12, cis-15 18:3. An inverse interaction was observed on the concentration of trans-11 18:1 for which the increase obtained with LO was of a lower magnitude with HC as compared with LC. This FA is an intermediate in the biohydrogenation process of both cis-9, cis-12 18:2 and cis-9, cis-12, cis-15 18:3. A shift from the trans-11 to the trans-10 pathway of biohydrogenation has been observed with the HC in the current trial as well as in previous experiments (Griinari & Bauman, 1999). The production of FA intermediates of the trans-10 pathway has also been reported during the biohydrogenation of cis-9, cis-12, cis-15 18:3 (Lee & Jenkins, 2011). Destaillats et al. (2005) proposed a putative biohydrogenation pathway for cis-9, cis-12, cis-15 18:3 in which the hydrogenation process begins with a cis-12/trans-13 isomerisation. In the current study, despite chromatographic conditions that allowed identification of this specific isomer, cis-9, trans-13, cis-15 18:3 was not detected in milk fat. Consistent with the present study, Zened et al. (2011) showed no detectable concentration of cis-9, trans-13, cis-15 18:3 in ruminal content incubated in vitro with cis-9, cis-12, cis-15 18:3 supplement. However, these conclusions are in contradiction with previous reports showing the presence of cis-9, trans-13, cis-15 18:3 in ruminant meat (Plourde et al. 2007) and milk (Rego et al. 2009). Discrepancies between studies remain to be elucidated and could potentially be related to experimental models, interactions with basal diets, and methodological and/or chromatographic conditions. Nevertheless, the presence of trans-13 18:1 and cis-9, trans-13 18:2, which both increased following LO supplementation, confirm the existence of a trans-13 pathway of hydrogenation. Also, cis-9, trans-13 18:2 might have been produced endogenously in tissues by the Δ9-desaturation of trans-13 18:1 (Bichi et al. 2012). Other isomers of 18:2 with a trans-13 double bond are increased when cis-9, cis-12, cis15 18:3 is provided in the diet. Lerch et al. (2012) observed higher concentrations of cis-11, trans-13 18:2 and trans-11, trans-13 18:2 in milk fat when feeding lactating dairy cows with extruded linseed. The isomer trans-13, cis-15 18:2 is another potential intermediate in a trans-13 pathway of biohydrogenation (Destaillats et al. 2005). However, this conjugated 18:2 isomer remains to be identified in milk fat.
Linseed oil and biohydrogenation intermediates Among other 18:3 isomers, which could be intermediate in a trans-13 pathway, Lerch et al. (2012) identified cis-9, trans-11, trans-13 18:3 in bovine milk fat. However, the hydrogenation of this fully conjugated 18:3 isomer is more likely to produce trans-11, trans-13 18:2 which has been reported in ruminal content (Loor et al. 2004) and milk (Lerch et al. 2012) in dairy cows. The level of trans-14 18:1 in milk fat was about 60% higher than the level of trans-13 18:1, and the ratio of these two FA was affected neither by LO supplementation nor by level of concentrate (P > 0·10; data not shown). Unlike the ratio of trans-11 18:1 to trans-10 18:1, which was significantly reduced when changing from LC to HC diets (P < 0·01; data not shown), the ratio of trans-13 to trans-14 does not appear to be modified by the conditions of ruminal environment. Shingfield et al. (2010) integrated the data available in the literature on the biohydrogenation of cis-9, cis-12, cis-15 18:3, and suggested cis-12, trans-14 18:2 and trans-12, trans-14 18:2 as putative intermediates in the production of trans-14 18:1. In particular, results from other studies have shown an increase in trans-12, trans-14 18:2 (Gómes-Cortés et al. 2009; Lerch et al. 2012), cis-9, trans-14 18:2, and cis-12, trans-14 18:2 (Lerch et al. 2012) when diets of lactating ruminants were supplemented with extruded linseed. In the current trial, the high concentration of trans-14 18:1 observed when feeding LO (7·5 fold increase compared with NLO with the LC diets) also supports the existence of a trans-14 pathway of biohydrogenation of cis-9, cis-12, cis-15 18:3. The increase in milk fat content of trans-10, cis-12 18:2 and trans-10 18:1 when feeding LO in diets with a similar level of concentrate (LC or HC), or the higher trans-10 18:1 when increasing level of concentrate in diets with similar LO supplementation (NLO or LO) is in line with the lower milk fat content observed with these two treatments as reported previously under similar situations (Shingfield et al. 2010). However, higher milk yield compensated for the lower fat concentration so that total fat yield was not affected by LO, and even tended to be higher with HC diets. Lack of effect of LO on milk fat yield has been observed previously with similar levels of supplementation (Loor et al. 2005; Benchaar et al. 2012), but a decrease in milk fat content and yield has been observed with higher dietary supplies of linseed oil (Chilliard et al. 2009), and raw or extruded linseed (Akraim et al. 2007). In conclusion, results of the current experiment confirmed that biohydrogenation of cis-9, cis-12, cis-15 18:3 is dominated by the trans-11 pathway; for which the highest concentrations of intermediates have been found in milk fat (i.e. cis-9, trans-11, cis-15 18:3, trans-11, cis-15 18:2, cis-15 18:1, and trans-11 18:1). Moreover, the extent of production of these intermediates has been affected by the levels of concentrate in the diet, as shown by the interactions observed between treatments for milk fat content of trans11, cis-15 18:2 and trans-11 18:1. The identification and quantification of trans-13 18:1 and cis-9, trans-13 18:2 still offers some support to a trans-13
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pathway of ruminal biohydrogenation. However, adding linseed oil, high levels of concentrate, or both did not result in a shift toward a trans-13 biohydrogenation pathway. Finally, cis-9, trans-13, cis-15 18:3 has not been identified in our milk samples, which suggests that this specific biohydrogenation pathway is not initiated by a cis-12 to trans-13 isomerisation. This experiment was funded through Industrial Research Chair programme of Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada), with industry contributions from Dairy Farmers of Canada (Ottawa, ON, Canada), Novalait Inc. (Québec, QC, Canada), Valacta (Ste-Anne-de-Bellevue, QC, Canada), Fédération des Producteurs de Lait du Québec (Longueuil, QC, Canada), and Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (Québec, QC, Canada). The authors thank André Perreault and Philippe Cantin as well as the administrative staff of the Centre de Recherche en Sciences Animales de Deschambault (Québec, Canada) for the care provided to cows during the trial. The authors are also grateful to Gabrielle StPierre and Micheline Gingras from the Département des Sciences Animales, Université Laval for their assistance in samplings and laboratory analyses.
References Akraim F, Nicot MC, Juaneda P & Enjalbert F 2007 Conjugated linolenic acid (CLnA), conjugated linoleic acid (CLA) and other biohydrogenation intermediates in plasma and milk fat of cows fed raw or extruded linseed. Animal 1 835–843 Allen MS 2000 Effects of diet on short-term regulation of feed intake by lactating dairy cattle. Journal of Dairy Science 83 1598–1624 AOAC International 2012 Official Methods of Analysis, 19th edition. Gaithersburg, MD: Association of Official Analytical Chemists International Bauman DE & Griinari JM 2001 Regulation and nutritional manipulation of milk fat: low-fat milk syndrome. Livestock Production Science 70 15–29 Benchaar C, Romero-Pérez GA, Chouinard PY, Hassanat F, Eugene M, Petit HV & Côrtes C 2012 Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition. Journal of Dairy Science 95 4578–4590 Bichi E, Toral PG, Hervás G, Gómez-Córtes P, Juárez M & de la Fuente MA 2012 Inhibition of Δ9-desaturase activity with sterculic acid: effect on the endogenous synthesis of cis-9 18:1 and cis-9, trans-11 18:2 in dairy sheep. Journal of Dairy Science 95 5242–5252 Canadian Council on Animal Care 1993 Guide to Care and Use of Experimental Animals, Vol. 1 (Eds ED Offert, BM Cross & AA McWilliam). Ottawa, ON, Canada Chilliard Y, Martin C, Rouel J & Doreau M 2009 Milk fatty acid in dairy cows fed whole crude linseed, extruded linseed, or linseed oil, and their relationship with methane output. Journal of Dairy Science 92 5199– 5211 Chouinard PY, Lévesque J, Girard V & Brisson GJ 1997 Dietary soybeans extruded at different temperatures: milk composition and in situ fatty acid reactions. Journal of Dairy Science 80 2913–2924 Destaillats F, Trottier JP, Galvez JMG & Angers P 2005 Analysis of α-linolenic acid biohydrogenation intermediates in milk fat with emphasis on conjugated linolenic acids. Journal of Dairy Science 88 3231–3239 Emery RS 1978 Feeding for increased milk protein. Journal of Dairy Science 61 825–828
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Flachowsky G, Erdmann K, Hüther L, Jahreis G, Möckel P & Lebzien P 2006 Influence of roughage/concentrate ratio and linseed oil on the concentration of trans-fatty acids and conjugated linoleic acid in duodenal chyme and milk fat of late lactating cows. Archives of Animal Nutrition 60 501–511 Gómez-Cortés P, Tyburczy C, Brenna JT, Juárez M & de la Fuente MA 2009 Characterization of cis-9 trans-11 trans-15 C18:3 in milk fat by GC and covalent adduct chemical ionization tandem MS. Journal of Lipid Research 50 2412–2420 Griinari JM & Bauman DE 1999 Biosynthesis of conjugated linoleic acid and its incorporation into meat and milk in ruminants. In Advances in Conjugated Linoleic Acid Research, Vol 1, pp. 180–200 (Eds MP Yurawecz, MM Mossoba, JKG Kramer, MW Pariza, GJ Nelson). Champaign, IL, USA: AOCSPress Griinari JM, Dwyer DA, McGuire MA, Bauman DE, Palmquist DL & Nurmela KVV 1998 Trans-octadecanoic acids and milk fat depression in lactating dairy cows. Journal of Dairy Science 81 1251–1261 Griinari JM, Corl BA, Lacy SH, Chouinard PY, Nurmela KVV & Bauman DE 2000 Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by Δ9 desaturase. Journal of Nutrition 130 2285–2291 Harfoot CG 1981 Lipid metabolism in the rumen. In Lipid Metabolism in Ruminant Animals, pp. 21–55 (Ed. WW Christie). New York, NY, USA: Pergamon Press. Hurtaud C, Faucon F, Couvreur S & Peyraud JL 2010 Linear relationship between increasing amounts of extruded linseed in dairy cow diet and milk fatty acid composition and butter properties. Journal of Dairy Science 93 1429–1443 Kramer JKG, Hernandez M, Cruz-Hernandez C, Kraft J & Dugan MER 2008 Combining results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as demonstrated using Ag-ion SPE fractionation. Lipids 43 259–273 Lee YJ & Jenkins TC 2011 Biohydrogenation of linolenic acid to stearic acid by the rumen microbial population yields multiple intermediate conjugated diene isomers. Journal of Nutrition 141 1445–1450 Lerch S, Shingfield KJ, Ferlay A, Vanhatalo A & Chilliard Y 2012 Rapeseed or linseed in grass-based diets: effects on conjugated linoleic and conjugated linolenic acid isomers in milk fat from Holstein cows over 2 consecutive lactations. Journal of Dairy Science 95 7269–7287 Loor JJ, Ueda K, Ferlay A, Chilliard Y & Doreau M 2004 Biohydrogenation, duodenal flow, and intestinal digestibility of trans fatty acids and conjugated linoleic acids in response to dietary forage:concentrate ratio and linseed oil in dairy cows. Journal of Dairy Science 87 2472–2485
Loor JJ, Ferlay A, Ollier A, Doreau M & Chilliard Y 2005 Relationship among trans and conjugated fatty acids and bovine milk fat yield due to dietary concentrate and linseed oil. Journal of Dairy Science 88 726–740 Martin C, Rouel J, Jouany JP, Doreau M & Chilliard Y 2008 Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil. Journal of Animal Science 86 2642–2650 Petit HV 2010 Review: feed intake, milk production and milk composition of dairy cows fed flaxseed. Canadian Journal Animal Science 90 115–127 Petit HV, Dewhurst RJ, Scollan ND, Proulx JG, Khalid M, Haresign W, Twagiramungu H & Mann GE 2002 Milk production and composition, ovarian function, and prostaglandin secretion of dairy cows fed omega-3 fats. Journal of Dairy Science 85 889–899 Plourde M, Destaillats F, Chouinard PY & Angers P 2007 Conjugated α-linolenic acid isomers in bovine milk and muscle. Journal of Dairy Science 90 5269–5275 Precht D, Molkentin J, McGuire MA, McGuire MK & Jensen RG 2001 Overestimates of oleic and linoleic acid contents in materials containing trans fatty acids and analyzed with short packed gas chromatographic columns. Lipids 36 213–216 Rego OA, Alves SP, Antunes LMS, Rosa HJD, Alfaia CFM, Prates JAM, Cabrita ARJ, Fonseca AJM & Bessa RJB 2009 Rumen biohydrogenationderived fatty acids in milk fat from grazing dairy cows supplemented with rapeseed, sunflower, or linseed oils. Journal of Dairy Science 92 4530–4540 Shingfield KJ, Bernard L, Leroux C & Chilliard Y 2010 Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants. Animal 4 1140–1166 Sterk A, Johansson BEO, Taweel HZH, Murphy M, van Vuuren AM, Hendriks WH & Dijkstra J 2011 Effects of forage type, forage to concentrate ratio, and crushed linseed supplementation on milk fatty acid profile in lactating dairy cows. Journal of Dairy Science 94 6078–6091 Sukhija PS & Palmquist DL 1988 Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. Journal of Agricultural and Food Chemistry 36 1202–1206 Undersander D, Mertens DR & Thiex N 1993 Forage Analyses Procedures. Omaha, NE: National Forage Testing Association Van Soest PJ, Robertson JB & Lewis BA 1991 Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74 3583–3597 Zened A, Troegeler-Meynadier A, Nicot MC, Combes S, Cauquil L, Farizon Y & Enjalbert F 2011 Starch and oil in the donor cow diet and starch in substrate differently affect the in vitro ruminal biohydrogenation of linoleic and linolenic acids. Journal of Dairy Science 94 5634–5645
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Effect of feeding linseed oil in diets differing in forage to concentrate ratio: 1. Production performance and milk fat content of biohydrogenation intermediates of α-linolenic acid Leacady Saliba, Rachel Gervais, Yolaine Lebeuf and P. Yvan Chouinard* Département des sciences animales, Université Laval, Québec, Québec G1V 0A6, Canada Received 25 February 2013; accepted for publication 31 October 2013
To evaluate the interaction between the levels of dietary concentrate and linseed oil (LO) on milk fatty acid (FA) profile, 24 Holstein cows were used in a randomised complete block design based on days in milk, with a 2 × 2 factorial arrangement of treatments. Within each block, cows were fed one of four experimental diets containing 30% concentrate (LC) or 70% concentrate (HC), without LO (NLO) or with LO supplemented at 3% of dietary dry matter. Milk FA profiles were analysed with a special emphasis on the intermediates of the predominant trans-11, and a putative trans-13 pathways of ruminal biohydrogenation of cis-9, cis-12, cis-15 18:3. Feeding LO increased the concentrations of cis-9, cis-12, cis-15 18:3 and trans-11, cis-15 18:2 in milk fat, and these increases were of a higher magnitude when LO was added in HC as compared with LC diet (interaction of LO by concentrate). A treatment interaction was also observed for the level of trans-11 18:1 which was higher when feeding LO, but for which the increase was more pronounced with the LC as compared with HC diet. The concentrations of cis-15 18:1 and cis-9, trans-11, cis-15 18:3 were higher in cows fed LO, but feeding HC diets decreased milk fat content of cis-15 18:1 and a tendency for a decrease in cis-9, trans-11, cis15 18:3 was apparent. Feeding LO increased milk fat content of trans-13 18:1 and cis-9, trans-13 18:2, while the concentrations of these two isomers were not affected by the level of dietary concentrates. The isomer cis-9, trans-13, cis-15 18:3 has not been detected in any of the milk samples. In conclusion, interactions were observed between LO and dietary concentrates on the proportions of some intermediates of the trans-11 biohydrogenation pathway. The presence of trans-13 18:1 and cis-9, trans-13 18:2 supports the existence of a trans-13 pathway, but an 18:3 intermediate with a trans-13 double bond has not been identified. Keywords: Dairy cow, flaxseed, biohydrogenation, forage to concentrate ratio.
Full-fat linseed and linseed oil, which are sources of α-linolenic acid (cis-9, cis-12, cis-15 18:3), have been investigated for their effects on lactation (Petit, 2010) and reproductive (Petit et al. 2002) performance, FA profile and physical properties of milk fat (Hurtaud et al. 2010), and enteric methane emission (Martin et al. 2008) in dairy cows. When cows receive full-fat linseed or linseed oil, the ruminal biohydrogenation of cis-9, cis-12, cis-15 18:3 produces a large number of FA isomers (Loor et al. 2004; Flachowsky et al. 2006). These biohydrogenation intermediates can be absorbed and incorporated in milk fat of lactating animals, either directly, or following their metabolic transformation in body tissues (e.g. desaturation and elongation).
*For correspondence; e-mail: [email protected]
Dietary factors affecting ruminal biohydrogenation of linoleic acid (cis-9, cis-12 18:2) have been extensively evaluated (Bauman & Griinari, 2001). More specifically, increased production of trans-10 18:1 is observed when feeding high doses of unsaturated FA with low-fibre diets. However, less is known about the effects of diet on different pathways of ruminal biohydrogenation of cis-9, cis-12, cis15 18:3. The predominant pathway in the biohydrogenation of cis-9, cis-12, cis-15 18:3 involves an initial isomerisation where the cis-12 bond is converted to a trans-11 bond to produce cis-9, trans-11, cis-15 18:3 (Harfoot, 1981). The second and third steps consist of the hydrogenation of cis-9 and cis-15 bonds to produce trans-11, cis-15 18:2 and trans11 18:1, respectively. After absorption, trans-11 18:1 can be desaturated to cis-9, trans-11 18:2 by the action of the Δ9 desaturase (Griinari et al. 2000). Destaillats et al. (2005)
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Table 1. Ingredients and chemical composition of experimental diets (g 100/g dry matter) Treatment† NLO Pretreatment
LC
LO HC
LC
HC
Ingredient, g/100 g of dry matter Corn silage Grass/Legume silage‡ Grass hay Rolled barley Cracked corn Soybean meal Corn gluten meal Linseed oil Vitamins and minerals
28·6 23·4 6·9 11·9 11·5 11·0 4·5 — 2·2
Dry matter, g/100 g Crude protein Starch ADF NDF NEL, Mcal/kg dry matter
47·8 15·0 30·1 20·1 33·1 1·61
14:0 16:0 cis-9 16:1 18:0 cis-9 18:1 cis-11 18:1 cis-9, cis-12 18:2 cis-9, cis-12, cis-15 18:3 20:0 Total fatty acids
60·0 25·0 60·0 10·0 5·0 10·0 9·0 28·6 9·0 9·0 28·6 5·4 6·7 7·5 7·3 3·3 3·3 3·3 — — 3·0 2·0 2·0 2·0 Chemical composition, g/100 g of dry matter 45·8 62·9 45·9 15·4 15·3 14·9 13·2 37·5 10·5 29·0 16·3 29·1 45·3 27·3 45·9 1·43 1·56 1·54 Fatty acid profile, g/100 g of dry matter 0·12 0·11 0·11 4·18 4·48 5·00 0·07 0·07 0·08 0·50 0·50 1·52 2·34 4·25 7·72 0·20 0·20 0·42 8·29 12·26 11·84 7·31 4·89 19·56 0·15 0·14 0·16 23·16 26·89 46·43
25·0 5·0 28·6 25·0 8·1 3·3 3·0 2·0 64·5 15·6 35·6 18·4 25·8 1·65 0·09 5·46 0·07 1·55 9·86 0·46 16·55 16·98 0·15 51·15
† NLO = no linseed oil; LO = with linseed oil, LC = low concentrate; HC = high concentrate ‡ Grass/legume silage was constituted of 11% ladino clover, 34% timothy, and 55% bromegrass
proposed an alternative pathway in the biohydrogenation of cis-9, cis-12, cis-15 18:3 involving an initial isomerisation where the cis-12 bond is converted to a trans-13 bond, leading to the sequential production of cis-9, trans-13, cis-15 18:3, cis-9, trans-13 18:2 or trans-13, cis-15 18:2, and trans-13 18:1. As for several octadecenoic acids, trans-13 18:1 could be desaturated after absorption to produce cis-9, trans-13 18:2 (Destaillats et al. 2005). In support of the pathway proposed by Destaillats et al. (2005), few experiments have shown the presence of trace amounts of cis-9, trans-13, cis-15 18:3 in meat and milk fat from ruminants (Plourde et al. 2007; Rego et al. 2009). As opposed to cis-9, cis-12 18:2, the effects of diet on the different biohydrogenation pathways of cis-9, cis-12, cis-15 18:3 are not well documented. The objective of this experiment was therefore to evaluate the effect of feeding linseed oil in diets differing in forage to concentrate ratio (F:C) on milk yield and composition, and on milk FA profile. It was hypothesised that supplying linseed oil in different basal diets affects the relative importance of different pathways involved in ruminal biohydrogenation of
cis-9, cis-12, cis-15 18:3, resulting in varying concentrations of specific FA intermediates in milk fat.
Materials and methods Animals and diets The experiment was conducted at the Centre de Recherche en Sciences Animales de Deschambault (Deschambault, QC, Canada). All procedures involving dairy cows followed the regulations of the Canadian Council on Animal Care (1993), and were approved by the Université Laval animal care committee. Twenty-four lactating Holstein cows (637 ± 68 kg body weight; 168 ± 53 d in milk; 9 primiparous and 15 multiparous) housed in a tie-stall barn were fed a pretreatment total mixed ration based on grass/legume silage and corn silage (Table 1). Cows were then assigned to a randomised complete block design based on days in milk with a 2 × 2 factorial arrangement of treatments. Within each block, cows were fed one of four experimental diets
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containing 30% concentrate (LC) or 70% concentrate (HC), without linseed oil (NLO) or with linseed oil (LO) supplemented at 3% of dietary dry matter (Table 1). The treatment period lasted 4 weeks. Throughout the experiment, diets were offered to the cows once daily as a total mixed ration, and the amount of feed offered was adjusted to ensure 10% refusals. Free access to water was provided at all time. Sampling, measurements and analyses A first sampling and measurement phase was conducted during the last week of the pretreatment period (week 0), and a second during the last week of the treatment period (week 4). At each of those phases, cows were weighed for 3 consecutive days following the a.m. milking. Dry matter intake was recorded and feed samples were collected for 3 consecutive days and pooled by treatment. A first subsample was dried at 55 °C for 3 days to determine dry matter intake. All rations were analysed by wet chemistry (Dairy One Laboratory, Ithaca, NY, USA) according to the following methods: dry matter (method 2.2.1.1; National Forage Testing Association; Undersander et al. 1993), residual moisture (method 991.01; AOAC International, 2012), ADF (ANKOM Technology method 5: ADF in feeds – filter bag technique for A200; solutions as in method 973.18; AOAC International, 2012), NDF (ANKOM Technology method 6: NDF in feeds – filter bag technique for A200; solutions as in Van Soest et al. 1991), and starch (application note number 319; YSI Inc. Life Sciences, Yellow Springs, Ohio). After freeze drying and grinding through a 1-mm screen, FA profile of total mixed rations was determined. Fatty acids were directly transesterified, and FA methyl esters were extracted following the method described by Sukhija & Palmquist (1988) with modifications. In the modified procedure, benzene was replaced by toluene and incubation in 3 ml HCl 5% (v/v) was performed at 50 °C for 30 min and followed a prior incubation in 3 ml sodium methoxide (0·5 M in methanol) at 70 °C for 60 min. Cows were milked at 0700 and 1700 h. Milk was sampled, and milk yield was recorded for 3 consecutive days. Daily milk composites were made from p.m. and a.m. milk samples proportionally to milk yield. A first subsample was preserved with bronopol and kept at 4 °C before analyses of fat, protein, and lactose using a Foss Milkoscan 4000 (Foss Electric, Hillerød, Denmark) combined with a Bentley 2000 (Bentley Instruments, Chaska, MN) at Valacta (Dairy Production Centre of Expertise, Ste-Anne-deBellevue, Québec, Canada). A second subsample was stored at 20 °C without preservative for FA analysis. Before lipid extraction for FA analysis, milk samples were thawed in water at 36 °C for 30 min. Milk fat was then extracted and FA were methylated according to Chouinard et al. (1997). Milk FA profile was determined using a gas chromatograph (Agilent 7890A; Agilent Technologies, Santa Clara, CA, USA) equipped with a 100-m CP-Sil-88 capillary column (0·25 μm i.d., 0·20 μm film thickness; Agilent
Technologies Canada Inc., Mississauga, Canada), and a flame ionisation detector. In order to screen milk FA composition, a first temperature programme was used where, at the time of injection, the column temperature was 80 °C for 1 min., then ramped at 2 °C/min. to 215 °C and maintained for 21·5 min. Two additional isothermal temperature programmes (150 and 175 °C; Kramer et al. 2008) were used to separate isomers that were co-eluting in the first programme. For the separation and quantification of cis-9, trans-11, cis-15 18:3 and cis-9, trans-13, cis-15 18:3, a fourth temperature programme was used where at the time of sample injection, the column temperature was 120 °C for 180 min., then increased at 10 °C/min. to 220 °C and maintained isothermal for 20 min. (Plourde et al. 2007). For the separation and quantification of trans-13 18:1 and trans14 18:1, trans FA methyl esters were first isolated using silver ion-thin layer chromatography followed by a GC analysis of this fraction with a fifth programme at a low isothermal temperature of 120 °C (Kramer et al. 2008). Most FA peaks were identified and quantified using either a quantitative mixture or pure methyl ester standards (Larodan Fine Chemicals, Malmö, Sweden; Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada; Matreya LLC, Pleasant Gap, PA, USA; Nu Chek Prep, Elysian, MN, USA; Naturia, Sherbrooke, QC, Canada). Standards for isomers that were not available commercially were identified by order of elution according to Precht et al. (2001) and Kramer et al. (2008). Statistical analysis Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) with two models depending on type of data collected. The first model was used for body weight, dry matter intake, milk yield, and milk composition. This model included data collected during the last week of the pre-trial period as covariates, and also level of concentrate, oil supplementation, and their interaction as fixed effects. Block was included as a random effect. Because data on milk FA profile from the pre-trial period were not available, a second model was used to determine treatment effects on milk FA composition which included the fixed effects of level of concentrate, oil supplementation, and their interaction, and block as a random effect. Results are reported as least square means and standard errors of the means. Differences between treatments were declared at P 4 0·05, and tendencies from P > 0·05 to P < 0·10.
Results Dietary concentrations of crude protein were similar between treatments (Table 1). Both HC diets (NLO and LO) contained about 40% more dry matter, three times more starch, and 40% less ADF and NDF than both LC diets (NLO and LO). The HC diets which contained higher concentration of cracked corn and rolled barley had 40% more cis-9
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Table 2. Body weight, dry matter intake (DMI), milk yield, and milk composition in cows fed high (HC) or low (LC) concentrate diets without supplemental oil (NLO), or supplemented at 3% of dry matter with linseed oil (LO) Treatment NLO
LO
Item
LC
HC
Body weight, kg DMI, kg/d Milk yield, kg/d
648 21·5 25·4
663 25·6 32·2
Fat Protein Lactose
4·22 3·33 4·60
Fat Protein Lactose
1,046 836 1,174
P-value†
LC
654 19·6 26·9 Composition, g/100 g 3·76 4·07 3·59 3·12 4·72 4·58 Yield, kg/d 1,165 1,106 1,136 829 1,525 1,236
HC
SEM
LO
C
LO × C
653 23·9 34·1
6 0·8 1·2
0·75 0·03 0·12
0·22 < 0·01 < 0·01
0·21 0·90 0·87
3·37 3·42 4·80
0·09 0·03 0·04
< 0·01 < 0·01 0·40
< 0·01 < 0·01 < 0·01
0·18 0·50 0·18
1,160 1,160 1,640
48 40 58
0·54 0·82 0·11
0·07 < 0·01 < 0·01
0·46 0·68 0·64
† P-value for the effects of linseed oil (LO), concentrate (C), and the interaction of linseed oil by concentrate (LO × C)
Table 3. Fatty acid intake in cows fed high (HC) or low (LC) concentrate diets without supplemental oil (NLO), or supplemented at 3% of dry matter with linseed oil (LO) Treatment NLO
LO
P-value†
Fatty acid, g/day
LC
HC
LC
HC
SEM
LO
C
14:0 16:0 cis-9 16:1 18:0 cis-9 18:1 cis-11 18:1 cis-9, cis-12 18:2 cis-9, cis-12, cis-15 18:3 20:0 Total fatty acids
2·6 90·0 1·5 10·9 51·0 4·3 179·0 158·1 3·2 500·5
2·8 114·3 1·8 12·4 106·7 5·0 312·4 120·0 3·6 678·6
2·2 97·6 1·6 29·8 150·0 8·2 228·6 385·8 3·1 907·2
2·2 130·0 1·7 36·8 233·8 10·9 393·1 403·7 3·6 1215·3
0·1 4·6 0·1 1·4 8·4 0·4 13·8 15·1 0·1 43·4
< 0·01 0·01 0·65 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·73 < 0·01
0·13 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·46 < 0·01 < 0·01
LO × C 0·11 0·35 0·12 0·04 0·09 0·01 0·24 0·06 0·71 0·12
† P-value for the effects of linseed oil (LO), concentrate (C), and the interaction of linseed oil by concentrate (LO × C)
18:1 and cis-9, cis-12 18:2, and 19% less cis-9, cis-12, cis-15 18:3 compared with LC diets. The addition of LO doubled the concentration of total FA in the diet compared with NLO treatments. Moreover, adding LO to diets increased the proportions of 18:0, cis-9 18:1, and cis-9, cis-12, cis-15 18:3 by about 3-fold as compared with NLO. No interaction of oil by concentrate has been observed for animal performance and concentration of major milk constituents (Table 2). Final body weight after the 4-wk experimental period was similar among treatments. Feeding HC diets increased dry matter intake, milk yield, as well as protein and lactose content and yield (P < 0·01). Adding LO decreased dry matter intake and milk protein content, but increased intake of cis-9, cis-12 18:2 and cis-9, cis-12, cis-15 18:3 (Table 3). Fat content decreased with LO
supplementation, as well as with high concentrate diets (P < 0·01), while fat yield tended to be higher with HC compared with LC (P = 0·07). Feeding LO increased the concentration of cis-9, cis-12, cis-15 18:3 in milk fat (Table 4), and this increase was of a higher magnitude when LO was added in HC as compared with LC diet (interaction of LO by concentrate P < 0·01). The concentration of cis-9, cis-12 18:2 was not affected by LO, but was increased when feeding HC as compared with LC diets. A treatment interaction was observed for the level of trans11 18:1 which was increased by feeding LO, but for which the increase was more pronounced with the LC as compared with HC diets. Milk fat concentrations of trans-15 18:1 and trans-11, cis-15 18:2 were also higher in milk of cows fed LO,
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Table 4. Milk fat composition in cows fed high (HC) or low (LC) concentrate diets without supplemental oil (NLO), or supplemented at 3% of dry matter with linseed oil (LO) Treatment NLO Item, g/100 g Saturated 4:0 5:0 6:0 7:0 8:0 9:0 10:0 11:0 12:0 iso 13:0 anteiso 13:0 13:0 iso 14:0 14:0 iso 15:0 anteiso 15:0 15:0 iso 16:0 16:0 iso 17:0‡ anteiso 17:0§ 17:0 iso 18:0 18:0 19:0 20:0 22:0 24:0 Monounsaturated cis-9 10:1 trans 12:1¶ cis-9 12:1 cis-9 14:1 cis-11 14:1 trans 15:1¶ cis-9 15:1 trans-4 16:1 trans-5 16:1 trans-6-7 16:1 trans-8 16:1 trans-9 16:1 cis-7 16:1 + trans-11-12 16:1 cis-9 16:1 + trans-13 16:1 cis-11 16:1 cis-12 16:1 + trans-14 16:1 cis-13 16:1 cis-14 16:1 cis-7 17:1 cis-8 17:1 cis-9 17:1 trans-4 18:1 trans-5 18:1 trans-6-8 18:1
LO
P-value†
LC
HC
LC
HC
SEM
LO
C
LO × C
2·935 0·024 1·818 0·028 1·074 0·030 2·627 0·049 3·132 0·024 0·007 0·082 0·090 10·662 0·219 0·401 1·129 0·197 30·540 0·258 0·354 0·484 0·046 7·398 0·038 0·147 0·060 0·039
2·355 0·033 1·540 0·042 1·028 0·057 3·048 0·130 3·967 0·024 0·005 0·177 0·054 11·615 0·173 0·379 1·418 0·162 27·473 0·286 0·362 0·491 0·032 7·366 0·052 0·121 0·037 0·025
2·908 0·016 1·502 0·015 0·756 0·014 1·572 0·015 1·721 0·023 0·007 0·043 0·074 7·715 0·172 0·328 0·785 0·143 19·555 0·258 0·303 0·400 0·046 12·380 0·010 0·197 0·055 0·032
2·352 0·021 1·338 0·021 0·827 0·021 2·074 0·044 2·496 0·027 0·006 0·081 0·045 9·586 0·150 0·339 0·846 0·210 19·951 0·301 0·342 0·364 0·029 11·625 0·013 0·170 0·039 0·021
0·157 0·002 0·090 0·004 0·061 0·006 0·150 0·013 0·150 0·003 0·001 0·015 0·006 0·293 0·012 0·021 0·082 0·023 1·019 0·012 0·018 0·017 0·003 0·607 0·004 0·008 0·004 0·003
0·93 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·71 0·63 < 0·01 0·05 < 0·01 < 0·01 0·02 < 0·01 0·86 < 0·01 0·50 0·04 < 0·01 0·61 < 0·01 < 0·01 < 0·01 0·58 0·02
< 0·01 < 0·01 0·02 0·02 0·84 0·01 < 0·01 < 0·01 < 0·01 0·50 0·03 < 0·01 < 0·01 < 0·01 0·01 0·78 < 0·05 0·37 0·12 < 0·01 0·15 0·38 < 0·01 0·52 0·08 < 0·01 < 0·01 < 0·01
0·94 0·39 0·53 0·30 0·35 0·12 0·79 < 0·05 0·84 0·53 0·81 0·06 0·57 0·12 0·32 0·45 0·18 0·01 < 0·05 0·51 0·33 0·21 0·58 0·56 0·21 0·90 0·38 0·41
0·261 0·068 0·078 1·025 0·044 0·024 0·013 0·005 0·006 0·012 0·023 0·055 0·133 1·229 0·027 0·073 0·205 0·016 0·030 0·043 0·161 0·006 0·007 0·140
0·230 0·077 0·103 1·008 0·070 0·016 0·011 0·005 0·008 0·010 0·021 0·030 0·160 1·202 0·029 0·040 0·228 0·014 0·028 0·019 0·161 0·007 0·009 0·161
0·146 0·029 0·035 0·628 0·019 0·020 0·012 0·006 0·007 0·006 0·025 0·098 0·189 0·863 0·018 0·110 0·093 0·008 0·023 0·035 0·144 0·025 0·019 0·336
0·171 0·042 0·053 0·782 0·035 0·013 0·009 0·004 0·006 0·007 0·021 0·044 0·182 0·802 0·020 0·064 0·114 0·008 0·020 0·016 0·118 0·012 0·014 0·307
0·012 0·004 0·004 0·053 0·003 0·002 0·001 0·001 0·001 0·001 0·002 0·006 0·008 0·094 0·002 0·007 0·009 0·001 0·001 0·001 0·013 0·001 0·001 0·019
< 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·03 0·11 0·48 0·88 < 0·01 0·35 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·03 < 0·01 < 0·01 < 0·01
0·80 < 0·01 < 0·01 0·17 < 0·01 < 0·01 0·03 0·11 0·77 0·74 0·04 < 0·01 0·18 0·64 0·36 < 0·01 0·02 0·44 < 0·01 < 0·01 0·31 < 0·01 0·07 0·82
0·02 0·55 0·44 0·09 0·09 0·91 0·37 0·16 0·08 0·10 0·38 0·02 0·04 0·85 0·74 0·39 0·92 0·53 0·48 < 0·01 0·32 < 0·01 < 0·01 0·20
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87
Table 4. (Cont.) Treatment NLO
LO
P-value†
Item, g/100 g
LC
HC
LC
HC
SEM
LO
trans-9 18:1 trans-10 18:1 trans-11 18:1 trans-12 18:1 trans-13 18:1 trans-14 18:1 trans-15 18:1 trans-16 18:1 cis-6-8 18:1 cis-9 18:1†† cis-11 18:1 cis-12 18:1 cis-13 18:1 cis-14 18:1 cis-15 18:1 cis-9 20:1 cis-11 20:1 Nonconjugated 18:2 trans-9, trans-12 18:2 trans-8, cis-12 18:2 trans-8, cis-13 18:2 trans-9, cis-12 18:2 trans-11, cis-15 18:2 cis-9, trans-12 18:2 cis-9, trans-13 18:2 cis-9, cis-12 18:2 Conjugated 18:2 cis-9, trans-11 18:2‡‡ trans-10, cis-12 18:2 Other unsaturated cis-6, cis-9, cis-12 18:3 cis-9, cis-12, cis-15 18:3 cis-9, trans-13, cis-15 18:3 cis-9, trans-11, cis-15 18:3 cis-11, cis-14 20:2 cis-8, cis-11, cis-14 20:3 cis-11, cis-14, cis-17 20:3 cis-5, cis-8, cis-11, cis-14 20:4 cis-8, cis-11, cis-14, cis-17 20:4 cis-5, cis-8, cis-11, cis-14, cis-17 20:5 cis-13, cis-16 22:2 cis-7, cis-10, cis-13, cis-16 22:4 cis-4, cis-7, cis-10, cis-13, cis-16 22:5 cis-7, cis-10, cis-13, cis-16, cis-19 22:5 cis-4, cis-7, cis-10, cis-13, cis-16, cis-19 22:6 Other fatty acids Glycerol
0·117 0·123 1·130 0·122 0·074 0·116 0·212 0·192 0·151 13·183 0·352 0·169 0·079 0·036 0·036 0·096 0·018
0·127 0·182 0·637 0·177 0·110 0·170 0·166 0·216 0·116 14·271 0·741 0·230 0·086 0·033 0·023 0·094 0·021
0·288 0·306 2·176 0·563 0·526 0·879 0·456 0·719 0·164 19·283 0·849 0·634 0·129 0·100 0·085 0·131 0·027
0·236 0·562 1·002 0·498 0·464 0·741 0·556 0·634 0·082 19·711 0·804 0·410 0·116 0·066 0·064 0·141 0·027
0·016 0·066 0·106 0·032 0·035 0·058 0·036 0·034 0·012 0·755 0·048 0·025 0·012 0·007 0·007 0·007 0·003
< 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·34 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01
0·20 0·03 < 0·01 0·89 0·71 0·48 0·39 0·38 < 0·01 0·28 < 0·01 < 0·01 0·77 0·01 < 0·01 0·56 0·62
0·07 0·15 < 0·01 0·07 0·18 0·11 0·03 0·13 0·06 0·63 < 0·01 < 0·01 0·33 0·03 0·42 0·42 0·55
0·047 0·105 0·052 0·019 0·164 0·017 0·094 1·181
0·025 0·162 0·063 0·019 0·049 0·023 0·122 1·921
0·157 0·299 0·212 0·069 0·880 0·060 0·261 1·240
0·063 0·300 0·233 0·035 0·359 0·056 0·235 1·670
0·011 0·023 0·017 0·006 0·048 0·006 0·019 0·097
< 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·25
< 0·01 0·22 0·37 0·01 < 0·01 0·94 0·93 < 0·01
< 0·01 0·25 0·76 0·01 < 0·01 0·43 0·16 0·07
0·515 0·002
0·359 0·003
0·879 0·004
0·510 0·004
0·063 0·0005
< 0·01 0·04
< 0·01 0·23
0·11 0·17
0·026 0·377 nd 0·025 0·028 0·080 0·008 0·108 0·006 0·045 0·041 0·020 0·015 0·051 0·007 0·825 12·360
0·031 0·298 nd 0·021 0·027 0·109 0·007 0·146 0·007 0·034 0·021 0·029 0·016 0·055 0·009 0·718 12·224
0·019 0·463 nd 0·050 0·022 0·058 0·013 0·084 0·007 0·036 0·033 0·019 0·011 0·056 0·009 1·913 11·891
0·017 0·550 nd 0·037 0·019 0·063 0·010 0·084 0·008 0·037 0·020 0·019 0·017 0·060 0·008 1·419 11·851
< 0·01 < 0·01 — < 0·01 < 0·01 < 0·01 < 0·01 < 0·01 0·08 0·08 < 0·01 0·02 0·43 0·18 0·75 < 0·01 < 0·01
0·35 0·91 — 0·06 0·25 < 0·01 0·20 0·01 0·03 0·02 < 0·01 0·03 0·12 0·32 0·77 0·02 0·24
0·03 0·01 — 0·31 0·38 0·02 0·35 0·02 0·45 < 0·01 0·06 0·04 0·21 0·97 0·41 0·10 0·52
0·001 0·030 — 0·004 0·002 0·005 0·001 0·008 0·001 0·002 0·002 0·003 0·002 0·004 0·002 0·113 0·073
† P- value for the effects of linseed oil (LO), concentrate (C), and the interaction of linseed oil by concentrate (LO × C) ‡ Coelution with minor concentration of trans-10 16:1 § Coelution with minor concentration of cis-10 16:1 ¶ Position of the double bound was not confirmed †† Coelution with minor concentration of cis-10 18:1 ‡‡ Coelution with trans-7, cis-9 18:2
C
LO × C
88
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but the increase was of a lower magnitude when LO was added in LC compared with HC diets. Feeding LO increased milk fat content of trans-13 18:1 and cis-9, trans-13 18:2, while their concentrations were not affected by dietary concentrates (Table 4). The isomer cis-9, trans-13, cis-15 18:3 has not been detected in any of the milk samples. The concentrations of cis-15 18:1, cis-9, trans-11 18:2 and cis-9, trans-11, cis-15 18:3 were higher in milk of cows fed LO, but feeding HC diets decreased milk fat content of these three FA without interaction between dietary treatments. Dietary LO increased the concentrations of trans-10 18:1 and trans-10, cis-12 18:2 in milk fat; and the proportion of trans-10 18:1 was further increased by feeding HC as compared with LC. Finally, the level of milk 18:0 was increased by feeding LO, but was not affected by dietary concentrates.
Discussion Feeding LO, and supplying more concentrate, increased the dietary proportions of FA and starch, and therefore improved the energy density of the diets (Table 1). As observed in the current trial, Sterk et al. (2011) also reported that shifting from a high forage (65:35 F:C) to a high concentrate (35:65 F:C) diet increased dry matter intake and milk yield in dairy cows. The higher dry matter intake with HC diets is in agreement with a lower dietary NDF content as suggested by Allen (2000). The observed increase in milk yield with HC could then be explained by a greater intake of dry matter, with a higher nutrient density. Supplementing dairy diets with LO, at similar or lower concentrations than the 3% level used in the current trial, has been shown to have no effect on dry matter intake (Loor et al. 2005; Flachowsky et al. 2006; Benchaar et al. 2012). However, Chilliard et al. (2009) reported a lower dry matter intake with higher levels of LO supplementation (5·7% of dry matter) using corn silage based diets. Feeding HC diets, with higher starch content, increased milk protein concentration and yield. Sterk et al. (2011) observed similar results when supplying a greater amount of starch, a glucogenic precursor which has been shown by these authors to be correlated with milk protein synthesis. Also, in a review by Emery (1978), a positive correlation was established between milk protein concentration and energy intake when concentrate was substituted for forage. The effects observed in the current trial on milk composition are consistent with previously published results showing lower milk fat content when feeding high concentrate diets (Griinari et al. 1998; Sterk et al. 2011). However, the decrease in milk fat content was more than compensated by an increase in milk production, so that a tendency for a higher fat yield was observed with HC diets in the present experiment. Dietary addition of LO increased the intake of cis-9, cis-12, cis-15 18:3, and this increase tended to be of a higher
magnitude with HC as compared with LC (P = 0·06). These variations were reflected in milk fat, where concentrations of cis-9, cis-12, cis-15 18:3 were increased when cows received LO diets with the highest concentration being observed with HCLO. Similarly, feeding HC diets increased the intake of cis-9, cis-12 18:2 which explained the higher concentration of this essential FA in milk fat as compared with LC diets. Among the intermediates of ruminal biohydrogenation, the treatment interaction observed on the level of trans-11, cis-15 18:2, that was increased by feeding LO, but for which the increase was more pronounced with the HC as compared with LC, is consistent with a similar interaction observed in the supply of its precursor e.g., dietary cis-9, cis12, cis-15 18:3. An inverse interaction was observed on the concentration of trans-11 18:1 for which the increase obtained with LO was of a lower magnitude with HC as compared with LC. This FA is an intermediate in the biohydrogenation process of both cis-9, cis-12 18:2 and cis-9, cis-12, cis-15 18:3. A shift from the trans-11 to the trans-10 pathway of biohydrogenation has been observed with the HC in the current trial as well as in previous experiments (Griinari & Bauman, 1999). The production of FA intermediates of the trans-10 pathway has also been reported during the biohydrogenation of cis-9, cis-12, cis-15 18:3 (Lee & Jenkins, 2011). Destaillats et al. (2005) proposed a putative biohydrogenation pathway for cis-9, cis-12, cis-15 18:3 in which the hydrogenation process begins with a cis-12/trans-13 isomerisation. In the current study, despite chromatographic conditions that allowed identification of this specific isomer, cis-9, trans-13, cis-15 18:3 was not detected in milk fat. Consistent with the present study, Zened et al. (2011) showed no detectable concentration of cis-9, trans-13, cis-15 18:3 in ruminal content incubated in vitro with cis-9, cis-12, cis-15 18:3 supplement. However, these conclusions are in contradiction with previous reports showing the presence of cis-9, trans-13, cis-15 18:3 in ruminant meat (Plourde et al. 2007) and milk (Rego et al. 2009). Discrepancies between studies remain to be elucidated and could potentially be related to experimental models, interactions with basal diets, and methodological and/or chromatographic conditions. Nevertheless, the presence of trans-13 18:1 and cis-9, trans-13 18:2, which both increased following LO supplementation, confirm the existence of a trans-13 pathway of hydrogenation. Also, cis-9, trans-13 18:2 might have been produced endogenously in tissues by the Δ9-desaturation of trans-13 18:1 (Bichi et al. 2012). Other isomers of 18:2 with a trans-13 double bond are increased when cis-9, cis-12, cis15 18:3 is provided in the diet. Lerch et al. (2012) observed higher concentrations of cis-11, trans-13 18:2 and trans-11, trans-13 18:2 in milk fat when feeding lactating dairy cows with extruded linseed. The isomer trans-13, cis-15 18:2 is another potential intermediate in a trans-13 pathway of biohydrogenation (Destaillats et al. 2005). However, this conjugated 18:2 isomer remains to be identified in milk fat.
Linseed oil and biohydrogenation intermediates Among other 18:3 isomers, which could be intermediate in a trans-13 pathway, Lerch et al. (2012) identified cis-9, trans-11, trans-13 18:3 in bovine milk fat. However, the hydrogenation of this fully conjugated 18:3 isomer is more likely to produce trans-11, trans-13 18:2 which has been reported in ruminal content (Loor et al. 2004) and milk (Lerch et al. 2012) in dairy cows. The level of trans-14 18:1 in milk fat was about 60% higher than the level of trans-13 18:1, and the ratio of these two FA was affected neither by LO supplementation nor by level of concentrate (P > 0·10; data not shown). Unlike the ratio of trans-11 18:1 to trans-10 18:1, which was significantly reduced when changing from LC to HC diets (P < 0·01; data not shown), the ratio of trans-13 to trans-14 does not appear to be modified by the conditions of ruminal environment. Shingfield et al. (2010) integrated the data available in the literature on the biohydrogenation of cis-9, cis-12, cis-15 18:3, and suggested cis-12, trans-14 18:2 and trans-12, trans-14 18:2 as putative intermediates in the production of trans-14 18:1. In particular, results from other studies have shown an increase in trans-12, trans-14 18:2 (Gómes-Cortés et al. 2009; Lerch et al. 2012), cis-9, trans-14 18:2, and cis-12, trans-14 18:2 (Lerch et al. 2012) when diets of lactating ruminants were supplemented with extruded linseed. In the current trial, the high concentration of trans-14 18:1 observed when feeding LO (7·5 fold increase compared with NLO with the LC diets) also supports the existence of a trans-14 pathway of biohydrogenation of cis-9, cis-12, cis-15 18:3. The increase in milk fat content of trans-10, cis-12 18:2 and trans-10 18:1 when feeding LO in diets with a similar level of concentrate (LC or HC), or the higher trans-10 18:1 when increasing level of concentrate in diets with similar LO supplementation (NLO or LO) is in line with the lower milk fat content observed with these two treatments as reported previously under similar situations (Shingfield et al. 2010). However, higher milk yield compensated for the lower fat concentration so that total fat yield was not affected by LO, and even tended to be higher with HC diets. Lack of effect of LO on milk fat yield has been observed previously with similar levels of supplementation (Loor et al. 2005; Benchaar et al. 2012), but a decrease in milk fat content and yield has been observed with higher dietary supplies of linseed oil (Chilliard et al. 2009), and raw or extruded linseed (Akraim et al. 2007). In conclusion, results of the current experiment confirmed that biohydrogenation of cis-9, cis-12, cis-15 18:3 is dominated by the trans-11 pathway; for which the highest concentrations of intermediates have been found in milk fat (i.e. cis-9, trans-11, cis-15 18:3, trans-11, cis-15 18:2, cis-15 18:1, and trans-11 18:1). Moreover, the extent of production of these intermediates has been affected by the levels of concentrate in the diet, as shown by the interactions observed between treatments for milk fat content of trans11, cis-15 18:2 and trans-11 18:1. The identification and quantification of trans-13 18:1 and cis-9, trans-13 18:2 still offers some support to a trans-13
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pathway of ruminal biohydrogenation. However, adding linseed oil, high levels of concentrate, or both did not result in a shift toward a trans-13 biohydrogenation pathway. Finally, cis-9, trans-13, cis-15 18:3 has not been identified in our milk samples, which suggests that this specific biohydrogenation pathway is not initiated by a cis-12 to trans-13 isomerisation. This experiment was funded through Industrial Research Chair programme of Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada), with industry contributions from Dairy Farmers of Canada (Ottawa, ON, Canada), Novalait Inc. (Québec, QC, Canada), Valacta (Ste-Anne-de-Bellevue, QC, Canada), Fédération des Producteurs de Lait du Québec (Longueuil, QC, Canada), and Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (Québec, QC, Canada). The authors thank André Perreault and Philippe Cantin as well as the administrative staff of the Centre de Recherche en Sciences Animales de Deschambault (Québec, Canada) for the care provided to cows during the trial. The authors are also grateful to Gabrielle StPierre and Micheline Gingras from the Département des Sciences Animales, Université Laval for their assistance in samplings and laboratory analyses.
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